D-lactate is a chiral small molecule, and is the precursor for synthesis of a variety of chiral compounds, which is widely used in medicines, pesticides and chemical industries. D-polylactic acid, synthesized by using D-lactate as starting material, is bio-degradable, and is an ideal environment-friendly polymer material: it can replace materials like polyethylene, polypropylene and polystyrene to produce novel environment-friendly package material; it can replace traditional medical materials like silicone rubber and silicone oil to produce bone fixations and medical surgical sutures etc.; it can be used in textile industry, to produce comfortable, smooth, and glossy underwear. D-lactate can be used as starting material to synthesize lactate esters, which can be used in the production of spices, resin coatings, adhesives, and printing inks; it can be used to produce excellent herbicides: Puma Super, Whip Super (Hoechst, Germany), DuplosanR (BASF, Germany).
The preparation methods of D-lactate mainly include chiral resolution, enzyme conversion and fermentation. Since chiral resolution and enzyme conversion have problems like environmental pollution and high costs, currently the large-scale production of D-lactate are mostly based on microbial fermentations. The production of D-lactate through microbial fermentation becomes the primary method for producing D-lactate, due to its low cost and high safety of the product. The production of D-lactate with high optical purity is the major limitation for microbial fermentation.
Currently there are mainly two classes of strains used for lactate fermentation. The first is natural lactate-producing bacteria (mainly Lactobacillus), which are important commercial strains since they have good acid resistance and can produce D-lactate with high optical purity after genetic modifications (Demirici et al. 1992, J Indust Microbiol Biotechnol 11:23-28; Okano K et al 2009, Appl Environ Microbiol 75:462-467). The drawbacks of such producing bacteria is that they need complex medium during the fermentation, and cannot utilize pentose, which increase the production costs and the downstream isolation/purification costs. For such bacteria, generally calcium hydroxide or calcium carbonate is used as neutralizer for controlling fermentative pH, and the final fermentation product is calcium lactate. However, the separation of calcium lactate generally requires addition of sulfuric acid, and produces large amounts of calcium sulfate waste which is hard to handle. More importantly, D-lactate produced by such bacteria normally has a chiral purity lower than 98%, which is not qualified for the production of polylactic acid.
The other class is metabolically engineered bacteria, including Saccharomyces, Bacillus, Kluyveromyces, and Escherichia. These engineered bacterial stains are all improved in some aspects regarding the lactate production, such as expanding substrate-utilizing range, reducing the nutritional needs, or eliminating the need for plasmids or antibiotics (Bianchi et al. 2001, Appl Environ Microbiol 67:5621-5625; Grabar et al. 2006, Biotechnol Lett 28:1527-153; Stewart et al. 2013, Yeast 30:81-91). In microbial fermentation, E. coli is extensively investigated to obtain high-yielding strain due to its advantages, such as clear genetic background, easy to operate, easy to regulate, easy to culture, fast growth, capable of utilizing various carbon sources and being cultured in minimal mineral salt medium. Under anaerobic conditions, E. coli generally consumes saccharides or derivatives thereof, and produces formate, acetate, lactate, succinate, ethanol etc. during fermentation. The yield of wild-type E. coli of producing lactate is generally very low. Studies in recent years indicate that, genetic modifications to metabolic pathways of E. coli can obtain lactate high-yielding strains. Currently modification of E. coli to produce high optical purity D-lactate has become the research focus.
Zhou et al. obtained recombinant E. coli strain SZ63 by deleting genes encoding the pyruvate formate lyase (pflB), fumarate reductase (frdABCD), alcohol dehydrogenase (adhE) and acetate kinase (ackA) in E. coli W3110 (Zhou et al. 2003, Applied Environ Microbiol 69:399-407). This strain SZ63 can produce 48 g/L D-lactate in mineral salts mediums containing 5% glucose after fermentation for 168 hours, wherein the D-lactate yield was nearly 2 mol/mol and the chiral purity reached 99%. Zhou et al. used the same strategy to construct recombinant strain SZ186 in E. coli B strain, and obtain strain SZ194 by metabolic evolution. After fermented in mineral salts mediums containing 12% glucose for 72 hours, SZ194 produced 111 g/L D-lactate with a yield of nearly 2 mol/mol and the chiral purity of 95% (Zhou et al. 2005, Biotechnol lett 27:1891-1896; Zhou et al. 2006, Biotechnol Lett 28:663-670).
Grabar et al. (Grabar et al. 2006, Biotechnol Lett 28:1527-1535) further modified the strain SZ194, by deleting methylglyoxal synthase gene mgsA to obtain recombinant strain TG112, and further to obtain strain TG114 by metabolic evolution. This strain TG114 produced 118 g/L D-lactate after fermented in mineral salts mediums containing 12% glucose for 48 hours, with the optical purity of 99.9% and a yield of glucose of 0.98 g/g.
The above engineered E. coli all used potassium hydroxide as neutralizer. Potassium hydroxide costs more in industrial production, while ammonia and sodium hydroxide cost much less. Therefore, it is desired to produce E. coli strains that can use ammonia or sodium hydroxide as neutralizer to adjust pH during the fermentation.
In order to improve the D-lactate titer and/or yield of E. coli, it is desired to further modify the metabolic pathways of E. coli. Besides, to reduce the possibility of bacterial contamination in industrial production, more improvements were needed to optimize the E. coli physiological properties and the temperature for D-lactate fermentation needed to be increased.